SFRP1 reduction results in an increased sensitivity to TGF-β signaling

Background: Transforming growth factor (TGF)-beta plays a dual role during mammary gland development and tumorigenesis and has been shown to stimulate epithelial-mesenchymal transition (EMT) as well as cellular migration. The Wnt/beta-catenin pathway is also implicated in EMT and inappropriate activation of the Wnt/beta-catenin signaling pathway leads to the development of several human cancers, including breast cancer. Secreted frizzled-related protein 1 (SFRP1) antagonizes this pathway and loss of SFRP1 expression is frequently observed in breast tumors and breast cancer cell lines. We previously showed that when SFRP1 is knocked down in immortalized non-malignant mammary epithelial cells, the cells (TERT-siSFRP1) acquire characteristics associated with breast tumor initiating cells. The phenotypic and genotypic changes that occur in response to SFRP1 loss are consistent with EMT, including a substantial increase in the expression of ZEB2. Considering that ZEB2 has been shown to interact with mediators of TGF-beta signaling, we sought to determine whether TGF-beta signaling is altered in TERT-siSFRP1 cells. Methods: Luciferase reporter assays and real-time PCR analysis were employed to measure TGF-beta transcriptional targets. Western blot analysis was used to evaluate TGF-beta-mediated ERK1/2 phosphorylation. Migration chamber assays were utilized to quantify cellular migration. TERT-siSFRP1 cells were transfected with Stealth RNAi (TM) siRNA in order to knock-down the expression of ZEB2. Results: TERT-siSFRP1 cells exhibit a significant increase in both TGF-beta-mediated luciferase activity as well as TGF-beta transcriptional targets, including Integrin beta(3) and PAI-1. Phosphorylation of ERK1/2 is increased in TERT-siSFRP1 cells in response to enhanced TGF-beta signaling. Furthermore, when the TGF-beta pathway is blocked with a TGF-beta R antagonist (LY364947), cellular migration is significantly hindered. Finally, we found that when ZEB2 is knocked-down, there is a significant reduction in the expression of exogeneous and endogenous TGF-beta transcriptional targets and cellular migration is impeded. Conclusions: We demonstrate that down-regulation of SFRP1 renders mammary epithelial cells more sensitive to TGF-beta signaling which can be partially ameliorated by blocking the expression of ZEB2.